Structural change in alpha-chymotrypsin induced by complexation with alpha 1-antichymotrypsin as seen by enhanced sensitivity to proteolysis.

Both human neutrophil elastase (HNE) and free chymotrypsin (Chtr) proteolyze Chtr within the complex that Chtr forms with antichymotrypsin (ACT). As free Chtr is stable both to self-digestion and to digestion by HNE, these results are indicative of a stability and/or conformational change in Chtr that accompanies complex formation. As determined by both N-terminal sequence analysis and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS), the major initial sites of HNE cleavage of complexed Chtr are between gamma-chain residues A158/S159 and V188/S189. Significantly, this latter site is at the base of the S1 site that recognizes the P1 position of the serpin. A slower cleavage in the beta-chain between T139/G140 is also found. In addition, rACT is cleaved between residues V22/D23. The gamma-chain of complexed Chtr is also cleaved by free Chtr, but at different sites: L162/L163 and W172/G173. beta-Chain cleavages were also found between residues Q81/K82 and F114/S115. Cleavages similar to those described above were also found when Chtr was complexed with the L358F-rACT variant, but not for Chtr complexed with either of the smaller inhibitors bovine pancreatic trypsin inhibitor or turkey ovomucoid third domain, nor for the covalent adduct of Chtr with N-p-tosylphenylalanyl chloromethyl ketone. We conclude that the structural change in Chtr making it a proteinase substrate is coupled with the large conformational change in ACT following complex formation. Complexed Chtr is much less reactive toward proteolytic digestion in the presence of high salt than in its absence, in accord with the high-salt induced release of active enzyme from the Chtr.rACT complex and the suggestion that electrostatic interactions mediate the coupling of structural change between rACT and Chtr within the Chtr.rACT complex. Potential physiological consequences of this work are explored.